RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells
RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells
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DOI：
10.1016/j.jsbmb.2006.12.084
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发表时间：
2007-03-01
影响因子：
4.1
通讯作者：
Morris, H. A.
中科院分区：
文献类型：
--
作者：
Anderson, P. H.;Atkins, G. J.;Morris, H. A.
虽然1，25 D的局部合成已被假定为调节非肾细胞中细胞生长和分化的参数，但1，25 D在骨细胞中产生的生理作用仍不清楚。我们使用RNA干扰技术来抑制编码负责1，25 D合成的酶，25-羟基维生素D 1 α-羟化酶（CYP 27 B1）的mRNA。人骨肉瘤（HOS）细胞在正常生长条件下用25 D处理48小时之前，用针对CYP 27 B1的siRNA或非沉默RNA转染。在培养基中测量1.25 D的从头合成以及CYP 27 B1、骨钙素（OCN）和25-羟基维生素D 24-羟化酶（CYP 24）的mRNA水平。我们证明，HOS细胞表达CYP 27 B1 mRNA，代谢25 D，并分泌可检测水平的从头合成的1，25 D。通过RNAi沉默CYP 27 BI mRNA，导致1，25 D的产生受到抑制，随后OCN和CYP 24 mRNA表达降低。我们的研究结果表明，当地的1，25 D合成在骨微环境中具有旁分泌作用，这意味着维生素D代谢在人成骨细胞中代表了一个重要的生理途径，可能调节成骨细胞的成熟。皇冠版权所有（c）2006年出版的爱思唯尔有限公司保留所有权利。
Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1 alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48 h under normal growth conditions. De novo synthesis of 1.25D was measured in the media as well as rnRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B I mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.